Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Pat. No. 4,603,112, the disclosure of which patent is incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1986).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
The family Flaviviridae comprises approximately 60 arthropod-borne viruses that cause significant public health problems in both temperate and tropical regions of the world (Shope, 1980; Monath, 1986). Although some highly successful inactivated vaccines and live-attenuated vaccines have been developed against some of these agents, there has been a recent surge in the study of the molecular biology of flaviviruses in order to produce recombinant vaccines to the remaining viruses, most notably dengue (Brandt, 1988).
Flavivirus proteins are encoded by a single long translational open reading frame (ORF) present in the positive-strand genomic RNA. The genes encoding the structural proteins are found at the 5' end of the genome followed by the nonstructural glycoprotein NS1 and the remaining nonstructural proteins (Rice et al., 1985). The flavivirus virion contains an envelope glycoprotein, E, a membrane protein, M, and a capsid protein, C. In the case of Japanese encephalitis virus (JEV), virion preparations usually contain a small amount of the glycoprotein precursor to the membrane protein, prM (Mason et al., 1987). Within JEV-infected cells, on the other hand, the M protein is present almost exclusively as the higher molecular weight prM protein (Mason et al., 1987A; Shapiro et al., 1972).
Studies that have examined the protective effect of passively administered monoclonal antibodies (MAbs) specific for each of the three flavivirus glycoproteins (prM, E, NS1) have demonstrated that immunity to each of these antigens results in partial or complete protection from lethal viral challenge. Monoclonal antibodies to E can provide protection from infection by Japanese encephalitis virus (JEV) (Kimura-Kuroda et al., 1988; Mason et al., 1989), dengue type 2 virus (Kaufman et al., 1987) and yellow fever virus (YF) (Gould et al., 1986). In most cases, passive protection has been correlated with the ability of these E MAbs to neutralize the virus in vitro. Recently, Kaufman et al. (1989) have demonstrated that passive protection can also be produced with prMMAbs that exhibit weak or undetectable neutralizing activity in vitro. The ability of structural protein specific MAbs to protect animals from infection is consistent with the conventional hypothesis that structural protein antibodies attenuate viral infection by blocking virus binding to target cells. Passive protection experiments using MAbs to the NS1 protein of yellow fever virus (Schlesinger et al., 1985; Gould et al., 1986) and dengue type 2 virus (Henchal et al., 1988) have demonstrated that antibodies to this nonstructural glycoprotein can protect animals from lethal viral infection. Since these MAbs do not exhibit viral binding properties, their protection is presumably mediated by some less conventional mechanism of attenuation of viral infection (Gibson et al., 1988).
Additional support for the ability of NS1 immunity to protect the host from infection comes from direct immunization experiments in which NS1 purified from either yellow fever virus-infected cells (Schlesinger et al., 1985, 1986) or dengue type 2 virus-infected cells (Schlesinger et al., 1987) induced protective immunity from infection with the homologous virus.
Although significant progress has been made in deriving the primary structure of these three flavivirus glycoprotein antigens, less is known about their three-dimensional structure. The ability to produce properly folded, and possibly correctly assembled, forms of these antigens may be important for the production of effective recombinant vaccines. In the case of NS1-based vaccines, dimerization of NS1 (Winkler et al., 1988) may be required to elicit the maximum protective response. For the E protein, correct folding is probably required for eliciting a protective immune response since E protein antigens produced in E. coli (Mason et al., 1989) and the authentic E protein prepared under denaturing conditions (Wengler et al., 1989b) failed to induce neutralizing antibodies. Correct folding of the E protein may require the coordinated synthesis of the prM protein, since these proteins are found in heterodimers in the cell-associated forms of West Nile virus (Wengler et al., 1989a). The proper folding of E and the assembly of E and prM into viral particles may require the coordinated synthesis of the NS1 protein, which is coretained in an early compartment of the secretory apparatus along with immature forms of E in JEV-infected cells (Mason, 1989).
Attempts to produce recombinant flavivirus vaccines based on the flavivirus glycoproteins has met with some success, although protection in animal model systems has not always correlated with the predicted production of neutralizing antibodies (Bray et al., 1989; Deubel et al., 1988; Matsuura et al., 1989; Yasuda et al., 1990; Zhang et al., 1988; Zhao et al., 1987).
Yasuda et al. (1990) reported a vaccinia recombinant containing the region of JEV encoding 65 out of the 127 amino acids of C, all of prM, all of E, and 59 out of the 352 amino acids of NS1. Haishi et al. (1989) reported a vaccinia recombinant containing Japanese encephalitis sequences encoding 17 out of the 167 amino acids of prM, all of E and 57 out of the 352 amino acids of NS1.
Deubel et al. (1988) reported a vaccinia recombinant containing the dengue-2 coding sequences for all of C, all of prM, all of E and 16 out of the 352 amino acids of NS1.
Zhao et al. (1987) reported a vaccinia recombinant containing the dengue-4 coding sequences for all of C, all of prM, all of E, all of NS1, and all of NS2A. Bray et al. (1989) reported a series of vaccinia recombinants containing the dengue-4 coding sequences for (i) all of C, all of prM and 416 out of the 454 amino acids of E, (ii) 15 out of the 167 amino acids of prM and 416 out of the 454 amino acids of E, (iii) 18 amino acids of influenza A virus hemagglutinin and 416 out of the 454 amino acids of E, and (iv) 71 amino acids of respiratory syncytial virus G glycoprotein and 416 out of the 454 amino acids of E.
Despite these attempts to produce recombinant flavivirus vaccines, the proper expression of the JEV E protein by the vaccinia recombinants has not been satisfactorily obtained. Although Haishi et al. (1989) demonstrated cytoplasmic expression of JEV E protein by their vaccinia recombinant, the distribution was different from that observed in JEV infected cells. Yasuda et al. (1990) detected expression of JEV E protein by their vaccinia recombinant on the cell surface. Recombinant viruses that express the prM and E protein protected mice from approximately 10 LD.sub.50 of challenge virus. Yasuda et al. (1990) elicited anti-JEV immune responses as well as protection but reactivity to a panel of E specific monoclonal antibodies exhibited differences from the reactivity observed in JEV infected cells.
Dengue type 2 structural proteins have been expressed by recombinant vaccinia viruses (Deubel et al., 1988). Although these viruses induced the synthesis of the structural glycoprotein within infected cells, they neither elicited detectable anti-dengue immune responses nor protected monkeys from dengue infection. Several studies also have been completed on the expression of portions of the dengue type 4 structural and nonstructural proteins in vaccinia virus (Bray et al., 1989; Falgout et al., 1989; Zhao et al., 1987). Interestingly, a recombinant that contained the entire 5' end of the viral ORF extending from C to NS2A under the control of the P7.5 early-late promoter produced intracellular forms of prM, E, and NS1 but failed to induce the synthesis of extracellular forms of any of the structural proteins, even though a form of NS1 was released from cells infected with this recombinant virus (Bray et al., 1989; Zhao et al., 1987). Additional recombinant viruses that contained several forms of the dengue type 4 E gene with or without other structural protein genes have also been examined (Bray et al., 1989). Although several of these recombinant viruses were able to induce protection, they neither produced extracellular forms of E nor induced neutralizing antibodies.
It can thus be appreciated that provision of a flavivirus recombinant poxvirus which produces properly processed forms of flavivirus proteins, and of vaccines which provide protective immunity against flavivirus infections, would be a highly desirable advance over the current state of technology.